Isolation of specific proteins and cells from complex biological mixtures such as blood serves as the essential first step in analytical and preparative methods involved in a range of diagnostic, therapeutic and research applications. Differences in easily accessible physical properties such as size and density form the basis of quick, inexpensive separation methods such as electrophoretic separation of proteins or density gradient centrifugation of cells often used in isolating abundant targets. However, for a large class of low abundance target proteins and rare cells, such as antigen-specific antibodies or immune cells, binding affinity to the cognate antigen is their only distinguishing characteristic and forms the basis of current isolation methods for them.
Isolation of multiple antigen-specific antibodies is currently most commonly performed using serially performed binding, washing and elution steps with separate affinity matrices for each antigen. This is time intensive and can be can be prohibitively so for low abundance antibodies as it can take hours to capture sufficient amount of antibody in each binding step. Also each binding step involves unavoidable loss of sample, making these methods particularly difficult to apply to low availability clinical samples.
Antigen-specific cells are currently isolated using either capture on antigen-coated solid matrices, rosetting with antigen-coated red blood cells or magnetic particles followed by density gradient centrifugation or magnetic separation respectively, or by staining with fluorescent antigen and isolation by flow cytometric cell sorting (FACS) (1). The cost of instrumentation for first two methods can be relatively low but they need to be serially applied for each antigen of interest. Also since they usually work as batch processes with relatively small batches of cells, isolating rare populations maybe can be challenging with these methods. FACS based methods can sort multiple (up to six in commercial instruments) selected cell populations simultaneously. Also since flow cytometry works at the single-cell level it can be used to identify and isolate relatively rare cells as well and indeed has been used to isolate rare antigen-specific B cell populations (2). However, the instrument cost is high which makes the method inaccessible. Also serially evaluating the fluorescence of every single cell results in a throughput bottleneck and usually at most about 10,000 cells per second can be handled. For very rare populations (<0.1%), this results in a prohibitively large amount of time required to isolate a sufficient number of cells for downstream processing.